Binding studies of nNOS-active amphibian peptides and Ca2+ calmodulin, using negative ion electrospray ionisation mass spectrometry

Amphibian peptides which inhibit the formation of nitric oxide by neuronal nitric oxide synthase (nNOS) do so by binding to the protein cofactor, Ca2+calmodulin (Ca2+CaM). Complex formation between active peptides and Ca2+CaM has been demonstrated by negative ion electrospray ionisation mass spectrometry using an aqueous ammonium acetate buffer system. In all cases studied, the assemblies are formed with a 1:1:4 calmodulin/peptide/Ca2+ stoichiometry. In contrast, the complex involving the 20-residue binding domain of the plasma Ca2+ pump C20W (LRRGQILWFRGLNRIQTQIK-OH) with CaM has been shown by previous two-dimensional nuclear magnetic resonance (2D NMR) studies to involve complexation of the C-terminal end of CaM. Under identical conditions to those used for the amphibian peptide study, the ESI complex between C20W and CaM shows specific 1:1:2 stoichiometry. Since complex formation with the studied amphibian peptides requires Ca2+CaM to contain its full complement of four Ca2+ ions, this indicates that the amphibian peptides require both ends of the CaM to effect complex formation. Charge-state analysis and an H/D exchange experiment (with caerin 1.8) suggest that complexation involves Ca2+CaM undergoing a conformational change to a more compact structure.     

Individually addressable microelectrode array for monitoring oxygen and nitric oxide release

We have fabricated a six individual addressable gold working electrode microarray. The device is wirebonded to an eight-pin DIL package that can be easily interconnected to an external multi-channel potentiostat. A polyion complex film coating on the electrode surface provides a suitable coating for the growth of cells. The responses of oxygen and nitric oxide were assessed on uncoated and coated devices using electroanalytical methods. The film coating reduced the diffusion current by approximately 20% in both cases. No changes in the electrochemical mechanism were observed. Simultaneous recordings were obtained for 2 h in the presence of the cells, thus the device is stable for the duration of the bioanalytical measurements. Measurements were conducted to study the simultaneous changes in oxygen and nitric oxide levels in cultured fibroblast cells in the presence of growth hormones that cause cell proliferation. Increases in oxygen consumption of the cells were coupled with increases in nitric oxide levels when in the presence of the growth hormones. Use of a biological detergent to cause an oxidative burst resulted in a large increase in the current for potentials set to detect nitric oxide and oxygen.     

Ugi products containing boronate esters

The Ugi reaction with formylphenylboronic acid derivatives as the carbonyl component proceeds to give the corresponding boron-containing dipeptide derivatives (Ugi products) in good yields.      

Phase composition of electrodeposited silver-indium alloys

The phase composition of electrochemically obtained Ag-In alloy coatings electrodeposited at different conditions was determined. With the increase in the current density, both indium content and heterogeneity of the deposited layers increase. The amount of the indium-richer phase increase as well. Before the thermal treatment, the phases Ag, Ag₃In, and In₄Ag₉ are observed in coatings with spatio-temporal structures. As a result of heating the new phase Ag₄In appears at temperatures above 500 °C and indium is oxidized up to In₂O₃ from the oxygen in the heating chamber. Up to 500 °C, the spatio-temporal structures are still visible. Probably they consist of both Ag-rich α-phase and one of the phases of the alloy system with small indium content, such as Ag₄In or Ag₃In.     

First regioselective cyclometalation reactions of cobalt in arylketones: C-H versus C-F activation

Phenyl ketones react smoothly under mild conditions with low valent cobalt(i) adducts to form new and stable five-membered cobaltacycles via C-H and C-F activation.     

Enantioselective synthesis of the dimeric pyranonaphthoquinone core of the cardinalins using a late-stage homocoupling strategy

The enantioselective synthesis of a dimeric pyranonaphthoquinone closely related to the cardinalins is described. Whilst attempts to effect a double Hauser-Kraus annulation of enone 5 were unsuccessful using both bis-phthalide 4 and bis-sulfone 21, a single annulation of cyanophthalide 28 with enone 5 furnished functionalised naphthalene 31. Suzuki-Miyaura homocoupling of the aryl triflate 29 derived from 31 effected a late-stage construction of the biaryl bond and facilitated access to the biaryl 3. Double stereoselective lactol reduction installed the 1,3-cis stereochemistry of the pyran rings and a final double oxidative demethylation step furnished model dimer 1, completing the enantioselective synthesis of a dimeric pyranonaphthoquinone bearing the core structure of cardinalin 3.     

Translational diffusion of macromolecules and nanoparticles modeled as non-overlapping bead arrays in an effective medium

There are three objectives to the present work. First, starting from a boundary element (BE) formulation of low Reynolds number hydrodynamics, model the translational diffusion of macromolecules modeled as an array of non-overlapping beads, and show how this approach is equivalent to previous formulations of "bead hydrodynamics" and under what conditions. Second, show how this approach can be improved upon by accounting for the variation in forces over the surfaces of individual beads and also extending the approach to a gel modeled as an effective medium, EM. Third, develop a "combined obstruction and hydrodynamic effect" model of the translational diffusion of irregularly shaped macromolecules in a gel. In one of the cases studied, the BE approach is shown to be equivalent to previous "bead model" formulations in which intersubunit hydrodynamic interaction is modeled using the Rotne-Prager tensor. A bead model that accounts for the variation in hydrodynamic stress forces over the individual bead surfaces is shown to be in best agreement with exact results for simple bead arrays made up of 2-4 subunits. The translational diffusion of rods, modeled as strings of from 2 to 100 touching beads in dilute gels is examined. Interpolation formulas valid over a range of gel concentrations and rod lengths are derived for the parallel and perpendicular components of the diffusion tensor as well as the orientationally averaged diffusion tensor. The EM model accounts for the long-range hydrodynamic interaction exerted by the gel support matrix on the diffusing particle of interest but does not account for the reduction in diffusion caused by the direct obstruction of the gel, or steric effect. Both effects are accounted for by writing the translational diffusion in a gel as the product of two terms representing long-range hydrodynamic interaction and steric effects. Finally, the diffusion of a 564 base pair DNA in a 2% agarose gel is examined and model results are compared to experiment (Pluen, A.; Netti, P. A.; Jain, R. K.; Berk, D. A. Biophys. J. 1999, 77, 542-552). For reasonable choices of model parameters, fair agreement between theory and experiment is achieved.     

Intramolecular vibrational excitation of unfolding reactions in ZnII-substituted and metal-free cytochromes c: activation enthalpies from integrated fluorescence stokes shift and line shape excitation profiles

We have employed continuous-wave fluorescence spectroscopy to observe the light-induced formation of partially unfolded states of Zn(II)-substituted and metal-free (or free-base) cytochrome c (ZnCytc and fbCytc, respectively). In these experiments, the intrinsic porphyrin chromophore provides a vibrational excitation to the protein structure via intramolecular vibrational redistribution of the excess vibronic energy above the first excited singlet state. As the excitation light source is tuned, the fluorescence spectrum of both systems exhibits steplike transitions of the integrated Stokes shift, vibronic structure, and line width that mark apparent activation enthalpy barriers for structural transitions of the protein from the native state to a set of at least three partially unfolded states. The vibronic structure of the ZnCytc spectrum reports the exchange of the Zn(II) ion's native H18 and M80 axial ligands with non-native ligands as the excitation wavenumber is scanned through the three barriers. The metal ion's axial ligands contribute substantially to the stability of ZnCytc; the activation enthalpies for the corresponding transitions in fbCytc are one-third of those in ZnCytc. A comparison of the present results from ZnCytc with those obtained previously with picosecond time-resolved methods [Lampa-Pastirk and Beck, J. Phys. Chem. B 2006, 110, 22971-22974] indicates that the vibrationally excited protein structure propagates along an unfolding pathway from the native state that specifically populates the three states in order of their activation enthalpies. The excitation-wavenumber profile of the fluorescence line width is markedly inconsistent with a Maxwell-Boltzmann distribution over the three states. These results contrast with the general expectation of the protein-folding funnel hypothesis that a distribution of intermediate structures should result from the diffusive propagation of a nonequilibrium protein structure.     

A mild, one-pot synthesis of 4-quinolones via sequential Pd-catalyzed amidation and base-promoted cyclization

A mild, one-pot synthesis of 4-quinolones is described. Under the optimal conditions, a variety of 2-substituted 4-quinolones were synthesized via sequential palladium-catalyzed amidation of 2'-bromoacetophenones followed by base-promoted intramolecular cyclization.     

Evaluation of high-field asymmetric waveform ion mobility spectrometry mass spectrometry for the analysis of the mycotoxin zearalenone

A high-field asymmetric waveform ion mobility spectrometry (FAIMS)-based method for the determination of the mycotoxin zearalenone (ZON) and its metabolites α-zearalenol (α-ZOL), β-zearalenol (β-ZOL), and β-zearalanol (β-ZAL), in a cornmeal (maize) matrix is described. Detection limits achieved using the FAIMS device coupled with electrospray ionization (ESI) and mass spectrometric (MS) detection are 0.4 ng mL⁻¹ for ZON and 3 ng mL⁻¹ for α-ZOL + β-ZOL, and β-ZAL. This represents a significant improvement when compared to detection limits determined using ESI-MS or ESI-tandem mass spectrometry (MSMS) analytical methods. The developed flow-injection (FIA)-ESI-FAIMS-MS method was applied to reference materials ERM-BC-716 and ERM-BC-717 certified for ZON and excellent agreement with the certified values was observed.